**3.1. History of the discovery of the morbillivirus receptor**

186 New Approaches to the Study of Marine Mammals

infection may cause a fatal blow.

important role in the transmission of the virus.

Denmark during the 1988 epizootic of PDV [48].

**3. SLAM, a receptor of morbillivirus** 

coast of Mauritania in Africa died suddenly. Morbilliviruses were isolated from the dead seals, although distemper-like lesions were not detected in the animals [40]. Hence, it remains unclear whether morbillivirus was the agent responsible for the dramatic deaths. The involvement of an algal bloom was also suggested as the primary cause [41]. In any case, when populations of many marine mammal species are decreasing, morbillivirus

The origin, precise mode of transmission, and maintenance reservoir of morbilliviruses causing die-off epidemics in marine mammals remain to be elucidated. Morbilliviruses proliferate in the infected animal for a short time after infection but do not persist in the host [4]. After being shed from the infected host, they do not survive long in the environment [4]. For the transmission of morbilliviruses, therefore, close contact between acutely infected and susceptible animals is required. In addition, because a morbillivirus infection results in lifelong immunity in the infected animal, when the virus is maintained in an animal population, a constant supply of new susceptible animals is needed. It has been calculated that the minimal population size for MV maintenance is approximately 300,000 individuals [42]. In the mass die-off of European seals in 1988, the most likely viral source was an infected seal population in the Arctic region, which moved southward and made contact with the population on the European coast. This hypothesis was based on the results of serologic studies using archival seal sera. PDV-specific antibodies were not observed in European seal sera before 1988, indicating that the population was naive and had not been previously exposed to the virus [43]. However, specific antibodies were detected in sera obtained from arctic seals long before 1998 [44,45]. In addition, alterations in the migration patterns of Arctic harp seal (*Phoca groenlandica*) populations were recorded. They were seen much farther south than usual in northern European waters in the year prior to the epizootic of the harp seal population [46]. The harp seal population is extremely large, with four million individuals in Canadian waters alone, which is sufficient to maintain morbillivirus circulating within the population. Subclinically or subacutely affected animals might play an

It should be noted that morbillivirus transmission sometimes occurs between marine and land mammals. As described above, the mass die-offs of Baikal seals and Caspian seals were caused by infection with CDV [28,30,32]. The most likely source of infection was land animals infected with CDV, because outbreaks were common among the numerous feral and domestic dogs around the lake [47]. Accidental infection in the opposite direction was also reported. A farmed mink population fed infected seal meat was infected with PDV in

The characteristics of SLAM have been extensively studied in humans. SLAM (CD150) is a type I transmembrane protein, and there are many members of the SLAM family including

**2.2. Transmission and maintenance reservoir of marine morbilliviruses** 

The human CD46 molecule was first identified as a cellular receptor for Edmonston vaccine strains of MV [59,60]. The Edmonston strain was isolated from the blood and throat washings of a child with measles using primary human kidney cells in 1954 [61]. It was later adapted to chick embryo fibroblasts and is being used as an attenuated vaccine [62]. This strain grows well in many cell lines, such as Vero cells, and has become the most extensively studied MV strain in the laboratory. However, because CD46, a complement-regulatory molecule, is expressed on all human nucleated cells, its ubiquitous distribution cannot explain the lymphoid tropism of MV. At present, CD46 is thought to be a specific receptor of the Edmonston strain, which is presumed to acquire the ability to use CD46 by adapting to cultured human kidney cells.

On the other hand, many wild-type strains have been isolated from clinical samples using the marmoset B cell line (B95a) [63], but they do not grow on many CD46+ cell lines. In order to identify the receptor for wild-type MV, functional expression cloning of a cDNA library of B95a cells was carried out using the VSV pseudotype system. SLAM was shown to be a cellular receptor for wild-type MV [18]. CDV and RPV were also shown to use canine and bovine SLAMs for entry into host cells [19]. Thus, SLAM is thought to be the major receptor for wild-type morbillviruses.

Recently, Nectin 4, a cellular adhesion junction molecule, has been identified as the third receptor for MV in polarized epithelial cells [64,65]. Infection experiments in monkeys showed that MV initially targets SLAM-positive immune cells such as alveolar macrophages, dendritic cells, and lymphocytes, and later the viral infection spreads to the epithelial cells of the trachea, lungs, oral cavity, pharynx, or intestines, which are SLAMnegative cells [66,67]. Another infection experiment using epithelial cell receptor-blind MV, demonstrated that the mutant MV inoculated intranasally to monkeys shows virulence and infectivity toward lymphoid tissues, although the virus cannot cross the airway epithelium and cannot be shed in the air [68]. The molecule forms tight junctions on polarized epithelial cells and was shown to function as the receptor for effectively releasing MV to the apical side of epithelial cells [69,70]. This explains why MV is highly contagious. Thus, the wildtype MV posesses two types of receptor, SLAM for entry and propagation and Nectin 4 for viral release into the air.

Host-Virus Specificity of the Morbillivirus Receptor, SLAM, in Marine Mammals:

SP: signal peptide, TM: transmembrane region, V: immunoglobulin V-like domain, C2: immunoglobulin C2-like domain. Closed squares: ITSM and ITSM-like motifs, The *N*-linked glycosylation site and cysteine residues are

**Figure 1.** Schematic drawings of domain (a) and primary structure (b) of marine mammal SLAMs.

Phylogenetic trees based on SLAM and the morbillivirus H protein were constructed using the maximum-likelihood (ML) and Bayesian methods. In the phylogeny of SLAMs (Figure 2(a)), each taxonomic group, including primates (humans, chimpanzees, rhesus monkeys, and marmosets), cetaceans (Pacific white-sided dolphins and killer whales), artiodactyls (cows, buffalo, sheep, and goats), pinnipeds (spotted seals and walruses), and rodents (mice and rats), was monophyletic with a 100% ML bootstrap probability (BP) and a 1.00 Bayesian posterior probability (BPP). Manatee and elephant SLAMs, dog and pinipped SLAMs, and cetacean and artiodactyl SLAMs formed single clades, each with a 100% BP and a 1.00 BPP

Morbillivirus phylogeny based on MV H protein sequences reflected the host grouping, except for MV. CDV (dogs, Baikal seals) and PDV (seals), and PMV (porpoises) and DMV (dolphins), respectively, formed single clades each with 100% BP and 1.00 BPP support (Figure 2(b)). The monophyletic lineage of MV (human) (100% BP and 1.00 BPP) was within the grouping of ruminant viruses, RPV (cow) and PPRV (sheep and goat), with 100% BP and 1.00 BPP. These phylogenetic trees indicated that SLAMs and viral H proteins roughly coevolved. However, the monophyletic lineage of MV and the ruminant viruses RPV and PPRV suggested that human MV may have originated from ancestral RPV in cattle by acquiring a binding affinity for human SLAM, as proposed in a previous report based on the

*3.2.2. Phylogenetic analysis based on SLAM and morbillivirus H proteins* 

indicated by arrows and triangles, respectively.

value, respectively.

morbillivirus P gene [9]

Risk Assessment of Infection Based on Three-Dimensional Models 189
